Impact of moderate exercise on ovarian blood flow and early embryonic outcomes in mares.

The advent of embryo transfer has allowed horses to continue to train and compete during the breeding season. However, the associated stress of exercise may be detrimental to reproduction. The objectives of this study were to evaluate differing exercise protocols on reproductive blood flow and embryonic outcomes in mares. Light-horse mares were randomized into control (n = 4), partial-exercised (n = 6), and full-exercised (n = 6) groups. Partial-exercised mares were moderately exercised 30 min daily during the periovulatory period and rested after ovulation for 7 d. Full-exercised mares were exercised for 30 min daily throughout the reproductive cycle. Mares were artificially inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 post ovulation. Blood flow through both ovarian arteries and vascular perfusion of the wall of the preovulatory follicle were examined by color Doppler ultrasonography. Results indicated exercise induced greater serum cortisol concentrations (P < 0.05). Embryo recovery rates were reduced in exercised (20/46, 43%) compared with control (14/21, 67%) mares (P < 0.10). When examined separately, embryo recovery rates for partial-exercised (11/25, 44%) and full-exercised (9/21, 43%) mares were not significantly different. Additionally, fewer quality Grade 1 embryos were recovered from partial-exercised mares compared with both control and full-exercised mares (P < 0.05). Blood flow through both ovarian arteries was greater in both exercised groups in the days leading up to ovulation (P < 0.05). However, vascular perfusion of the wall of the preovulatory follicle on the day before ovulation was less in both partial-exercised (45.9 ± 3.0%) and full-exercised (44.8 ± 3.4%) mares vs. control (54.9 ± 3.6%; P < 0.05). In exercised mares, vascular perfusion of the follicle wall was greater when an embryo was recovered (P < 0.01). No differences were found in follicle ovulatory diameter among exercised and non-exercised mares. When groups were combined, follicle diameter was greater when an embryo was recovered (44.9 ± 1.0 mm) compared with an unsuccessful embryo recovery attempt (42.8 ± 0.7 mm; P < 0.05). In conclusion, these data demonstrated that exercise increased ovarian arterial blood flow leading up to ovulation and decreased vascular perfusion of the wall of the preovulatory follicle. Mares given rest the day after ovulation up until an embryo collection attempt did not improve embryo recovery rates.

Exercise affects both ovarian follicular dynamics and hormone concentrations in mares.

The objectives were to evaluate the effects of exercise on ovarian folliculogenesis and related hormones in mares. Mares (n = 11) were randomly assigned into a control (non-exercised) or treatment (exercised) group. Treatment mares (n = 5) were moderately exercised for 30 min, 6 d/wk. All mares underwent daily transrectal ultrasonographic examinations and ovarian follicles > 6 mm were measured. Blood samples were collected during the first (Cycle 1) and last (Cycle 4) cycle, and serum concentrations of cortisol, LH, and FSH were determined. Mean cortisol concentrations were elevated (P < 0.05) in exercised mares, 6.29 ± 0.22 compared with 5.62 ± 0.16 ng/dL (mean ± SEM), 30 min post exercise. There were no significant differences between groups in mean FSH concentrations; however, exercised mares had lower (17.3 ± 6.4 vs 41.1 ± 5.5 ng/mL; P < 0.05) peak LH concentrations. Furthermore, exercised mares experienced a longer (24.7 ± 0.8 vs 22.2 ± 0.8 d; P < 0.05) mean interovulatory interval for all cycles combined, fewer (P < 0.05) follicles 6 to 20 mm in diameter, and an increased (P < 0.05) number of follicles >20 mm following deviation. The dominant and largest subordinate follicle in exercised mares had a greater (P < 0.05) mean diameter on the day of deviation, suggesting delayed deviation. Exercised mares also tended (P = 0.06) to have an increased number of cycles with at least two dominant follicles compared to control (62 vs 36%, respectively), indicating a decreased ability of the largest follicle to assert dominance. Under the conditions of this study, moderately exercising mares induced higher cortisol concentrations, lowered peak LH concentrations, and altered ovarian follicular dynamics.

Advances in recombinant gonadotropin production for use in bovine superovulation.

Bovine ovarian hyperstimulation is a process that currently relies on pituitary-derived follicle stimulating hormone (FSH) to facilitate the maturation of multiple follicles to achieve dominance and eventual ovulation. The prevalence of this process, also called superovulation, has more than doubled in the past 10 years, but the efficiency of recovered transferable embryos has remained low at ~6 per collection. The use of pituitary-derived products presents other problems including contamination from other hormones, inconsistencies within and among batches, and the possibility of the spread of disease-transmitting agents. Recombinant gonadotropins have been engineered to yield varieties of FSH and luteinizing hormone from a myriad of heterologous hosts with the resulting products demonstrating various levels of biological activity. Research has also been devoted to alternative delivery methods to reduce the frequency of injections required in current superovulatory protocols. Together, recombinant gonadotropins and alternative delivery approaches potentially provide an economical alternative to the use of pituitary-derived products.

Efficacy of a commercially available post-thaw bovine semen sexing kit in both single-ovulating and hyperstimulated cows.

Currently, there are few inexpensive, reliable, effective methods for commercially separating X- and Y-chromosome bearing fresh and frozen bovine sperm. The objective of these experiments was to determine the efficacy of a commercially available post-thaw bovine semen sexing kit, HeiferPlus (HP) which claims to alter the sex ratio in favor of female calves following artificial insemination. Three trials included the insemination of hyperstimulated cows with Control or HP-treated semen, non-surgical embryo collection on Day 7, and a combined PCR/dot blot assay to determine embryo sex. Chi-square analysis showed that the Control group produced a greater proportion (p<0.0005) of female embryos than the HP group. There were no differences in the proportions of transferable compared with degenerate embryos or in number of ovulations, embryos, and unfertilized ova collected from Control compared with HP groups. When treatments were combined, one of the two bulls used in the hyperstimulation studies produced an overall greater proportion of females (p<0.05), suggesting a bull effect. Another trial involved the insemination of cows synchronized via OvSynch((R)) with fetal sexing via ultrasonography. Results of these studies indicated that HP semen sexing kit did not alter the sex ratio in favor of females in either hyperstimulated or single-ovulating cows; however, potential bull effects may be further evaluated to understand the capacity of HP with semen from specific bulls. Additionally, perhaps the sex of the surviving embryo can be manipulated by the maternal side, through ovarian, hormonal, oviductal, or uterine influences.

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium.

BACKGROUND: Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes. RESULTS: Analysis of the cell cycle stages revealed that 91.2 +/- 0.2% of confluent cells were at the G0/G1 phase and 54.1 +/- 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors. CONCLUSIONS: Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.

Effect of thoracotomy and lung resection on exercise capacity in patients with lung cancer.

BACKGROUND: Resection is the treatment of choice for lung cancer, but may cause impaired cardiopulmonary function with an adverse effect on quality of life. Few studies have considered the effects of thoracotomy alone on lung function, and whether the operation itself can impair subsequent exercise capacity. METHODS: Patients being considered for lung resection (n = 106) underwent full static and dynamic pulmonary function testing which was repeated 3-6 months after surgery (n = 53). RESULTS: Thoracotomy alone (n = 13) produced a reduction in forced expiratory volume in one second (FEV1; mean (SE) 2.10 (0.16) versus 1.87 (0.15) l; p<0.05). Wedge resection (n = 13) produced a non-significant reduction in total lung capacity (TLC) only. Lobectomy (n = 14) reduced forced vital capacity (FVC), TLC, and carbon monoxide transfer factor but exercise capacity was unchanged. Only pneumonectomy (n = 13) reduced exercise capacity by 28% (PVO2 23.9 (1.5) versus 17.2 (1.7) ml/min/kg; difference (95% CI) 6.72 (3.15 to 10.28); p<0.01) and three patients changed from a cardiac limitation to exercise before pneumonectomy to pulmonary limitation afterwards. CONCLUSIONS: Neither thoracotomy alone nor limited lung resection has a significant effect on exercise capacity. Only pneumonectomy is associated with impaired exercise performance, and then perhaps not as much as might be expected.

Relationship between follicular development and the decline in the follicle-stimulating hormone surge in heifers.

Experiment 1 was conducted to determine whether progesterone affects the pattern of the FSH surge or follicular development associated with a follicular wave in heifers. On Day 7 (Day 0 = ovulation), heifers were allocated into a group receiving prostaglandin F2alpha (PGF2alpha; n = 6) or a control group (n = 5). Twenty-four hours later, all detectable follicles (>/= 2 mm) were ablated (Hour 0). Follicular development was monitored Hours 0, 3, 6, 9, 12, and 16, at 8-h intervals thereafter until Hour 112. To monitor FSH concentrations, blood was sampled at Hours -24, 0, 3, 6, 9, 12, and 16, and at 8-h intervals thereafter until Hour 104. There were no differences (p > 0.05) between the PGF2alpha-treated group and controls in the patterns of the FSH surge or follicular development. Experiment 2 tested the hypothesis that 3-mm follicles do not have FSH-suppressing capacity and that suppression increases as follicles grow beyond 5 mm. Twenty-four hours after an injection of PGF2alpha (Days 6-8), heifers were subjected to either ablation of follicles >/= 2 mm or ovariectomy. Intact heifers were allocated into four groups (n = 5) in which all follicles of the new wave were ablated upon reaching either 3, 5, or 7 mm or were not ablated (controls). Blood was sampled at 8-h intervals to monitor FSH and estradiol-17beta. Averaged over Hours 8-120, FSH concentrations (ng/ml) were higher (p < 0.05) in the ovariectomized (2.02 +/- 0.05) and the 3-mm groups (1.91 +/- 0.05) than in the 5-mm (1.52 +/- 0.05), 7-mm (1.35 +/- 0.04), and control groups (1.33 +/- 0.05); and estradiol concentrations (pg/ml) were lower (p < 0.05) in the ovariectomized group (0.19 +/- 0.03) than in the 3-mm (1.48 +/- 0. 16), 5-mm (1.56 +/- 0.15), 7-mm (2.22 +/- 0.27), and control groups (2.55 +/- 0.49). In conclusion, the presence of endogenous progesterone did not affect FSH patterns or follicular development. Follicles

Follicular and FSH dynamics in ewes with a history of high and low ovulation rates.

Daily transrectal ultrasound scanning and twice-daily blood sampling were used to monitor the temporal relationships between FSH concentrations and follicle development during complete interovulatory intervals for ewes in which the ovulation rate in each of the 2 previous years was high or low (> or = 3 and < or = 2 ovulations, respectively). Follicles that reached > or = 5 mm were used to define a follicular wave and were tracked retrospectively to 3 mm (emergence). The hypothesis that FSH surges (identified with a computer program) and follicular waves (retrospectively determined based on ultrasound scanning) are temporally associated was supported in both groups by the emergence of an anovulatory or ovulatory follicular wave near the peak of an FSH surge. Further support for the hypothesis was a significant increase in FSH concentrations before and a significant decrease after follicular-wave emergence in both groups independent of the identification of FSH surges. Ewes with a history of high ovulation rates had smaller follicles (anovulatory and ovulatory) and more ovulations, but the 2 groups were similar in the number of ovulatory follicular waves and associated FSH surges, number and characteristics of the FSH surges, and mean FSH concentrations per interovulatory interval. Surges of FSH were periodic (every 3 or 4 d) regardless of the ovulation-rate group or follicle response. In ewes with a low ovulation rate, the nonovulatory FSH surges were most frequently associated with emergence of detected anovulatory follicular waves. In ewes with a high ovulation rate, more FSH surges were not associated with a detected follicular wave, as defined, presumably because the largest follicle did not reach 5 mm. The results indicated that the factors resulting in a high ovulation rate were not exerted through circulatory patterns or concentrations of FSH but involved a shorter growth phase and smaller maximal diameter of follicles.

Effects of ultrasound-guided transvaginal follicular aspiration on oocyte recovery and hormonal profiles before and after GnRH treatment.

Endocrine changes and recovered oocytes were evaluated during 16 wk of ultrasound-guided transvaginal follicular aspiration (TVFA) and prior to and following administration of GnRH at the cessation of aspiration. Nonlactating previously aspirated (PAC, n = 4) and non-aspirated, (AC, n = 4) Holstein cows were subjected to 16 wk of twice-weekly aspiration. Four control cows (OAC) were aspirated 1 time only at the final TVFA session (wk 16). Jugular blood samples were collected from all cows during aspiration, before and after the final TVFA session, and during an 18-d period following cessation of aspiration. Ovarian activity was monitored in all cows after cessation of aspiration for 18 d. The PAC and AC cows averaged 3.4 +/- 1.2 (+/- SE) and 6.8 +/- 1.2 oocytes per session, respectively. Progesterone concentrations during TVFA did not differ between the PAC and AC (0.8 +/- 0.1 and 0.9 +/- 0.1 ng/mL, respectively). Progesterone concentration in OAC was 4.5 +/- 0.2 ng/mL before TVFA, while the PAC and AC averaged 0.5 +/- 0.2 and 0.3 +/- 0.2 ng/mL, respectively, at 16 wk. At Week 16 LH was 1.0 +/- 0.2 ng/mL and it increased to 7.5 +/- 0.1 ng/mL after GnRH treatment. The LH concentration before the final aspiration session was higher at peak amplitude in PAC than in AC groups and peak length was longer in OAC than in AC cows (P < 0.07). Between 18 and 24 h after the last aspiration there were more LH peaks and greater peak frequencies in PAC than in OAC cows (P < 0.07), and the interval between peaks was longer in PAC and AC cows (P < 0.10) than in OAC cows. Mean FSH concentrations were lower (P < 0.01) for OAC than for PAC and AC groups at 20 and 24 h after the last aspiration. Follicle numbers after GnRH varied most among treatment groups for follicles < 9 mm, with the PAC, AC and OAC averaging 5.1 +/- 1.0, 5.1 +/- 1.0, and 3.8 +/- 1.0 follicles/d, respectively. Progesterone concentrations increased to 1.1 +/- 0.3 ng/mL in PAC cows and 2.5 +/- 0.3 and 3.4 +/- 0.3 ng/mL in AC and OAC groups, respectively, during the 18-d period. These results suggest that long-term TVFA affects progesterone, LH and FSH profiles and ovarian dynamics in cows.

Tuberculous pericarditis presenting as pericardial tamponade.

We present one of the last cases from the British Military Hospital in Hong Kong. A 30 year old woman with pericardial tuberculosis and tamponade is described.

Effectiveness of Menuzo's B2 medium with buffalo rat liver cells for development of in vitro matured/in vitro fertilized bovine oocytes.

PURPOSE: The effectiveness of two culture media on the development of bovine embryos in a buffalo rat liver (BRL) coculture system was investigated. METHODS: Bovine oocytes were matured and fertilized in vitro, then cocultured, 25 per well, for 7 days in 500 microliters of modified M199 or modified Menuzo's B2 medium over a BRL cell monolayer at 39 degrees C in an atmosphere of 5% CO2 in air. Medium 199 was modified by the addition of 10% of (v/v) fetal bovine serum (FBS), 9 mg/ml bovine serum albumin, 2 mM glycine, 1 mM alanine, and 0.1 mM nonessential amino acids (NEAA). Menuzo's B2 medium was modified by the addition of 10% (v/v) FBS, 1 mM alanine, and 0.1 mM NEAA. RESULTS: Modified Menuzo's B2 medium improved embryo development to the morula or blastocyst stage compared to modified M199 (121/353, 34.3%, versus 99/362, 27.3%, P < 0.05). CONCLUSIONS: These results suggest that Menuzo's B2 medium with modifications in a BRL coculture system can provide a significant benefit for culture of early bovine embryos over the traditional use of medium 199.

Functional interrelationships between follicles greater than 4 mm and the follicle-stimulating hormone surge in heifers.

The interrelationships between the FSH surge that initiates a follicular wave and the follicles in the wave were examined in heifers. In experiment 1, > or = 5-mm follicles were ablated 5 days after ovulation and heifers (n = 6/group) received a total dosage of 0, 37.5, 75, or 150 units of porcine FSH. Half of the FSH dosage was administered 24 h after ablation followed by the other half 12 h later. Blood samples were taken after the initial FSH injection for FSH assay, and ovaries were examined daily with ultrasound to monitor follicle growth. There were progressively higher FSH concentrations at the mean peak (8 h after initial injection in all groups) as the dosage increased (interaction of dose and time; p < 0.001). Compared to values in controls, the highest dosage (150 units) approximately doubled the number of 5- and 6-mm follicles; this then progressed into a 4- to 7-fold increase in the number of 7- and 8-mm follicles. In experiment 2, either all (controls; n = 6), two (n = 11), one (n = 6), or zero (n = 6) follicles of the first wave of an estrous cycle were retained and the remaining were ablated upon reaching 5 mm. Scanning and blood sampling were performed every 8 h for 72 h after the initial ablation. Mean FSH concentrations during 0 to 72 h decreased (p < 0.004) as the number of retained follicles increased. In heifers in the one-follicle group, the randomly chosen 5-mm follicle developed the characteristics of a dominant follicle. The following conclusions were made: 1) the number of follicles that advanced into a follicular wave was increased by exaggerating the height of the FSH surge, 2) all > or = 5-mm follicles of a wave contributed to the declining portion of the FSH surge, and 3) any 5-mm follicles at the emergence of a wave were capable of becoming the dominant follicle.

Stress fracture of the sternum: an unusual injury?

Stress fracture of the sternum is a rare condition which presents as acute anterior chest pain after repetitive upper-body exercise. Two case reports are presented and it is postulated that this is an often underdiagnosed condition which should be considered in the differential diagnosis of acute chest pain in the athlete. Awareness of the injury together with meticulous clinical examination supported by good quality radiographs or isotope bone scan may lead to an increase in the diagnosis of this injury.

Nuclear transfer in the bovine using microinjected donor embryos: assessment of development and deoxyribonucleic acid detection frequency.

Bovine embryos that had been microinjected with DNA were examined for their potential use as donor embryos in nuclear transfer. Donor embryos were obtained from oocytes collected by transvaginal oocyte aspiration, matured and fertilized in vitro, microinjected with a murine whey acidic protein-human protein C genomic DNA construct, and cultured in vitro on liver cells of buffalo rat (Rattus norvegicus). Blastomeres from these embryos were transferred into enucleated bovine oocytes received from an abattoir by electrofusion at 40 h postmaturation. Following 7 d of culture, the developmental stage was recorded, and resulting embryos were prepared for analysis by polymerase chain reaction. Embryos that were derived from microinjected donor embryos did not differ from control donor embryos (11 vs. 8.6%) in development to the morula and blastocyst stage. Of the biopsies from 20 microinjected donor embryos, 19 were positive for the injected DNA. Of 37 embryos developing normally, only 12 (32.4%) were positive for the injected DNA. These results indicate that microinjected embryos can be successfully used in a nuclear transfer program to produce additional viable embryos and that these embryos may be reliably screened for the transgene for transfer to recipients.

In vitro embryo production after microinjection and ovarian dynamics following transvaginal follicular oocyte aspiration.

Ultrasound-guided transvaginal follicular aspiration of oocytes from live cows combined with IVM, IVF and in vitro culture (IVC) is a procedure for producing preimplantation-stage bovine embryos and a source of oocytes for pronuclear microinjection of DNA for producing transgenic cattle. This experiment was designed to compare in vitro embryo development rates between oocytes derived from transvaginal follicular aspiration and those obtained from cows at slaughter. Nine cows were subject to a twice-weekly aspiration. Oocytes were aspirated with a 5 MHz ultrasound transducer packaged in a vaginal probe equipped with a dorsal-mounted needle guide (16-ga). All visible follicles (>2 mm) were punctured with a 17-ga, 55-cm needle at each aspiration session and the contents removed under vacuum suction. Oocytes underwent IVM/IVF/IVC. Microinjection of DNA was performed during the pronuclear stage of development, and the zygotes were co-cultured on Buffalo Rat Liver (BRL) cells in modified M199 at 39 degrees C in 5% CO2 and air. After 7 d in culture, embryos were removed and scored for development. A Chi-square analysis was used to compare transvaginal follicular-derived oocytes (microinjected and not) and slaughterhouse-derived, matured in transit oocytes (SHDMT; microinjected and not). Nonmicroinjected embryos resulting from IVF of transvaginal aspiration-derived oocytes developed to blastocysts at a higher rate than SHDMT oocytes (40.0 vs 30.8%; P < 0.05). There was no difference in development rates between the microinjected groups (aspiration = 15.9% vs SHDMT = 12.8%). Higher proportions of the embryos generated from the aspirated oocytes were of excellent or good quality following culture (P < 0.05). In the present experiments the effects of microinjection may overshadow some effects of ova source, but transvaginal follicular aspiration may provide a more consistent, synchronous population of oocytes than those derived from commercial slaughter house sources for use with in vitro systems.

Tracheobronchial injury in blunt and penetrating chest trauma.

Ten patients were seen in Northern Syria with tracheobronchial injury from June 1986 to July 1988. Eight were male; and five were children. Blunt trauma was the cause of rupture in five and penetrating trauma in five. Nine patients had associated injuries. In seven, the diagnosis was made within 24 h. The seven patients who had surgery were well at last follow-up, as was a child with a main bronchial tear who was treated conservatively. Two men died without having surgery, one of respiratory failure and sepsis and the other of hemorrhagic shock. The group's mean age was 17.5 years. The average hospital stay was six days (eight for survivors), and the follow-up period was seven months. The clinical presentations and outcome stress the essential role of early chest x-ray and bronchoscopy, as well as a high index of suspicion.

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos.

The effect of DNA microinjection at various times after in vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11 h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15 h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p < 0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p < 0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19 h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p < 0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.